This invention relates to a combination of interleukin-2 (IL-2) and/or interferon.beta. (IFN-.beta.) and tumor necrosis factor (TNF) and the use of this combination as an anti-tumor therapeutic agent.
IL-2, a lymphokine which is produced by normal peripheral blood lymphocytes and induces proliferation of antigen or mitogen stimulated T cells after exposure to plant lectins, antigens, or other stimuli, was first described by Morgan, D. A., et at., Science (1976), 193:1007-1008. It is now recognized that in addition to the growth factor properties of IL-2, IL-2 modulates a variety of functions of immune system cells in vitro and in vivo.
IL-2 was initially made by cultivating human peripheral blood lymphocytes (PBL) or other IL-2-producing cell lines. See, for example, U.S. Pat. No. 4,401,756. Recombinant DNA technology has provided an alternative to PBLs and cell lines for producing IL-2. Taniguchi, T. et al., Nature (1983), 302:305-310 and Devos, R., Nucleic Acids Research (1983), 11:4307-4323 have reported cloning the human IL-2 gene and expressing it in microorganisms.
U.S. Pat. No. 4,518,584 describes and claims muteins of IL-2 in which the cysteine normally occuring at position 125 of the wild-type or native molecule has been replaced with a neutral amino acid, such as serine alanine. Copending U.S. application Ser. No. 810,656 filed Dec. 17, 1985, now discloses and claims an oxidationresistant mutein such as IL-2 which is biologically active wherein each methionine residue of the protein from which the mutein is derived which methionine is susceptible to chloramine T or peroxide oxidation is replaced with a conservative amino acid such as alanine. These IL-2 muteins possess the biological activity of native IL-2. U.S. Pat. Nos. 4,530,787 and 4,569,790 disclose and claim methods for purifying recombinant native IL-2 and meteins thereof, as well as the purified form of IL-2.
U.S. Pat. No. 4,604,377 discloses an IL-2 composition suitable for reconstituting in a pharmaceutically acceptable aqueous vehicle composed of oxidized microbially produced recombinant IL-2. The IL-2 is noted as useful in combination with cytotoxic chemotherapy or irradition or surgery in the treatment of malignant or premalignant diseases in a direct therapeutic or adjuvant setting or in combination with other immune-modulating drugs, lymphokines (e.g., IL-1IL-3, CSF-1 and IFNs) naturally occurring or inducible anticellular toxins in treating malignant diseases.
Various therapeutic applications of human IL-2 have been investigated and reported by S. Rosenberg and colleagues (see Mule et al., Science (1984), 225:1487 and S. Rosenberg et al., New England Journal of Medicine (1985), 313:1485-1492, for example).
Interferons (IFN) constitute a group of naturally occurring proteins that are known to exhibit anti-viral, anti-tumor and immunoregulatory behavior. Two types of IFN have been identified based on differences in their observed biological properties and molecular structures: Type I and Type II. Beta-interferon (IFN-.beta. ) is a Type I IFN that can be induced in fibroblasts by viral challenge and contains about 165 amino acids. IFN-.alpha. is also a Type I IFN inducible in leukocytes, and IFN.gamma. is a Type II IFN that is induced in lymphocytes in response to specific mitogenic stimuli and contains 146 amino acids.
Human IFN-.beta. may be produced by recombinant DNA technology, as described, for example, in EP 28,033 published June 6, 1981 to Sugano, et al. and U.K. 2,063,882 published June 10, 1981 to Revel, et al. Additionally, the IFN-.beta. may be a mutein in which amino acids not essential to biological activity are deleted or replaced with other amino acids to increase stability, as described by U.S. Pat. No. 4,588,585, the disclosure of which is incorporated herein by reference. Mouse IFN-.beta. may also be produced by recombinant DNA technology.
After Paucker et al., Virology, 17:324-334 (1962) showed that IFN suppressed the growth rate of mouse L cells, many investigators have studied treatment of mouse L cells with IFN and inhibition of tumor cell proliferation by IFN. See, e.g. Borden, E. C., Ann. Intern. Med., 91:472-479 (1979).
Tumor necrosis factor (TNF) was first described by Carswell et al., PNAS (USA) (1975), 72:3666-3670 as an endotoxin-induced serum factor which causes necrosis of chemically transformed tumor cells when growing in mice. Purified preparations of murine TNF have been tested against murine and human cell lines in vitro. K. Haranaka and N. Satomi, Japan J. Exp. Med. (1981), 51:191. In contrast to normal cells, tumor cell lines from both species were susceptible to the cytotoxic activity of the mouse TNF. Furthermore, the murine TNF was reported to be toxic against both human-and mouse-transplanted tumors in nude mice. See K. Haranaka et al., Int. J. Cancer (1984), 34:263-267. Human TNF is also known to be cytotoxic to neoplastic cells, and has been produced in recombinant form. See Pennica et al., Nature (1984), 312:724-729; Shirai et al., Nature (1985), 313:803-806; Wang et al., Science (1985), 228:149-154.
The cloning of rabbit TNF is disclosed in EP 146,026, published June 26, 1985 (Dainippon PHarmaceutical Co., Ltd.) and EP 148,311, published July 17, 1985 (Asahi Kasei Kogyo Kabushiki). The cloning of human TNF having 151 and 155 amino acids (2 and 6 less than the native form) is disclosed in EP 155,549, published Sept. 25, 1985 (Dainippon Pharmaceutical Co., Ltd.), and human TNF having 155 amino acids is disclosed in EP 158,286, published Oct. 16, 1985 (Asahi Kasei Kogyo Kabushiki Kaisha) and corresponding GB 2,158,829A, published Nov. 20, 1985. The cloning of mature TNF (157 amino acids) and various modified forms (muteins) thereof is disclosed in EP 168,214, published Jan. 15, 1986 (Genentech) and PCT US85/01921, filed Oct. 3, 1985, published Apr., 1986 (Cetus Corporation). The latter, PCT 85/01921 corresponds to U.S. Ser. No. 760,661 filed July 30, 1985, now U.S. Pat. No. 4,677,063, the disclosure of which is incorporated herein by reference.
Combination chemotherapy using two or more anti-cancer drugs to treat malignant tumors in humans is currently in use in research and in the clinic. The anti-cancer drugs may be antimetabolites, alkylating agents, antibiotics, general poisons, etc. Combinations of drugs are administered in an attempt to obtain a synergistic cytotoxic effect on most cancers, e.g., carcinomas, melanomas, lymphomas and sarcomas, and to reduce or eliminate emergence of drug-resistant cells and to reduce side effects to each drug.
It is known that Type I and Type II interferons may be combined to produce a synergistic biological effect. See, for example, Fleishmann, W. R., Cancer Res. (1982), 42:869-875 and DeClercq, E., et al., Cancer Letters (1982), 15:223-228 (mouse IFNs), and European Patent Publ. 107,498 published May 2, 1984 (human IFN-.gamma. and IFN-.alpha. or -.beta.).
U.S. Pat. No. 4,518,584 to Mark et al. (Cetus Corporation) discloses the combination of IL-2 muteins with gamma-interferon, B cell growth factor, and IL-1. In addition, it has been disclosed that IL-2 may be used with IFN-.gamma. to treat tumor-bearing hosts with synergistic results (European Patent Publ. 149,551 published July 24, 1985 (Genentech) and German Patent Publication 3411184 published Oct. 31, 1985 (Deut Roten Kreuzes)) or with augmentation of natural killer activity (Svedersky et al., J. Immunol. (1984), 133:714-718 and Shalaby et al., J. Interferon Res. (1985), 5:571-581.) Lopez-Botet et al., Eur. J. Immunol. (1984), 14:1137-1141 reported, however, that IL-2 and IFN-.gamma. are not sufficient in combination to induce natural killer-like activity in human T cell clones. It is also known from Dempsey et al., J. Immun. (1982), 129:2504-2510 that the combination of IFN-.alpha. and IL-2 is more effective than IFN-.alpha. or IL-2 alone in causing natural killer cell activation.
Lymphotoxin and TNF were once thought to be synomymous, but Stone Wolff et al., J. Exp. Medl ., 159:828-843 (1984) has shown that they are not the same protein. Lymphotoxin has a molecular weight of 60,000-70,000 daltons, whereas TNF has a lower molecular weight. EP 131,789 published Jan. 23, 1985 (Sloan-Kettering) discloses the synergistic effect of lymphotoxin and IFN-.gamma. to treat tumors in mice. Williamson et al., Proc. Natl. Acad. Sci. (USA) 50:5397-5401 (1983) discloses the in vivo effects of human lymphotoxin and human IFN. Others have published on the combined activity of lymphotoxin and antitumor drugs or interferons. See Williams et al., J. Immunol., 130:518-520 (1983), Matsunaga et al., Cancer Letters, 20:21-28 (1983) and Papermaster et al., Human lymphokines, Khan et al., ed., p. 459-477 (June 30, 1982).
Dr. Talmadge of the Preclinical Screening Lab., BRMP has reported in 1986 the augmented effect of using TNF and IFN-65 to treat metastatic disease in mice. U.S. Pat. No. 4,650,674 issued Mar. 17, 1987, filed Dec. 3, 1984 (Genentech) discloses the synergistic effect of TNF and IFN to treat various tumors. EP 170,843, published June 20, 1985 (Boehringer Ingelheim) discloses the synergistic effect of TNF and IFN-.alpha., .beta. and/or .gamma. on cancerous growth, particularly mixtures containing TNF and IFN-.gamma.. See also Matthews et al., Chem. Abs. 92:108513h (1980), which discloses injecting rabbits with BCG and endotoxin to induce TNF and IFN in vivo, and Buessow et al.,Leukemia Research, 8:801-811 (1984), which discloses augmenting the cellmediated tumoricidal activity of the HL-60 cell line using IFN-.alpha..